Department of Neurology, Research Institute of Surgery, Daping Hospital, Third Military Medical University of Chinese PLA, Chongqing   400042, China

Tackle Key Project of Chongqing Municipal Science and Technology Ministry, No. 7830*  

Abstract
BACKGROUND: Mesenchymal stem cells (MSCs) are capable of differentiating into a variety of tissues and exhibit low immunogenicity.
OBJECTIVE: To investigate isolation and in vitro cultivation methods of human cord blood MSCs, to observe expression of neural stem cell (NSC) marker mRNA under induction, and to detect tumori-genicity in animals.
DESIGN, TIME AND SETTING: A cell biological, in vitro trial and a randomized, controlled, in vivo experiment were performed at the Department of Neurology, Daping Hospital at the Third Military Medical University of Chinese PLA from August 2006 to May 2008.
MATERIALS: Umbilical cord blood was collected from full-term-delivery fetus at the Department of Gynecology and Obstetrics of Daping Hospital, China. Eighteen BALB/C nu/nu nude mice were randomly assigned to three groups: back subcutaneous, cervical subcutaneous, and control, with   6 mice in each group.  
METHODS: Monocytes were isolated from heparinized human cord blood samples by density gra-dient centrifugation and then adherent cultivated in vitro to obtain MSC clones. After the cord blood MSCs were cultured for 7 days with nerve growth factor and retinoic acid to induce differentiation into NSCs, the cells (adjusted density of 1 × 107/mL) were prepared into cell suspension. In the back subcutaneous and cervical subcutaneous groups, nude mice were hypodermically injected with a 0.5-mL cell suspension into the back and cervical regions, respectively. In the control group, nude mice received a subcutaneous injection of 0.5 mL physiological saline into the back or cervical re-gions, respectively.
MAIN OUTCOME MEASURES: Cellular morphology was observed by inverted microscopy, cul-tured cord blood MSCs were examined by flow cytometry, expression of nestin and musashi-1 mRNA was detected by reverse-transcriptase polymerase chain reaction prior to and after induction, and tumorigenicity following cord blood MSC transplantation was assayed by hematoxylin-eosin staining.
RESULTS: Following adherent cultivation, the majority of cord blood monocytes became rhombic and strongly expressed CD29, but not CD34, CD11a, or CD11b. These results supported previously known characteristics of cord blood MSCs. Following differentiation induction, nestin and musashi-1 were expressed on the surface of NSCs, exhibiting strongest expression at 48 hours, and subse-quently reducing expression. Cultured cord blood MSCs were not tumorigenic in the nude mice. Cellular morphology displayed no malignant changes between the control and subcutaneous groups.
CONCLUSION: MSCs can be isolated from human cord blood, efficiently expanded under culture conditions, differentiated into NSCs following induction, and display no tumorigenicity in nude mice.
Key Words: cord blood mesenchymal stem cells; neural stem cells; induced differentiation; nestin; tumorigenicity

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